In the present study, we combined experimental manipulation of host density across two contrasting environments with assessment of host physiology and life-history in a natural population of great tits to understand causes of variation in Plasmodium infection. The key and novel findings of this study were, firstly, that manipulation of host density increases P. circumflexum infection, but not infection with P. relictum; and secondly; that host antioxidants and ROM physiology show species-specific patterns in relation to infection, and thirdly, that ROM increases with Plasmodium parasitaemia.
Avian malaria, ecology and demography
Host density was found to be the only ecological and demographic parameter of importance for P. circumflexum infection. This link was absent for infection with P.relictum. This suggests that population density reduces the bird’s ability to resist infection by P. circumflexum, the more virulent of the two Plasmodium species . Possibly, this is via more intense competition over resources or it could be mediated via higher levels of testosterone or stress hormones in high density areas which have been shown to supress the immune system . Alternatively, the vectors transmitting P. circumflexum either aggregate where hosts are abundant or that vectors carrying the parasite are able to infect several birds, whereas the vector transmitting P.relictum may be more widely distributed or only feed on one host at one point in time [33–35] but see .
In Wytham Woods; a woodland in the proximity of Bagley, recent work on great tits and the closely related sympatric host species, blue tits (Cyanistes caeruleus), have shown that the two Plasmodium species have pronounced differences in their spatial distributions and impacts on both hosts species [7, 32]. While P. circumflexum infections exhibit pronounced spatial structuring that is stable over years in both host species; P. relictum infections are effectively randomly distributed in space (Lachish et al. in press). Likewise, P. circumflexum infections are linked with reduced survival, particularly during the acute stage of infection ; whereas P. relictum infections are associated with reproductive costs . Hence the species-specific relationships between ecological factors and parasite prevalence detected in the present study are not unexpected. Moreover, in Wytham woods it was shown that territory size was linked to host density i.e., the larger territory the lower density. This estimate of host density did not explain patterns of Plasmodium infection in the conspecific blue tits, Cyanistes caeruleus. Similar to the present study, however, a positive association was found between large-scale breeding density (i.e., measured as number of nests within a 1500 m radius) and Leucocytozoons prevalence and parasitaemia in nestling eagle owls (Bubo bubo) . In temperate regions Plasmodium species show a bimodal peak in infection, one in autumn and one in spring, and the spring peak (measured here) is most often a relapse from the autumn infection gained in the previous year [31, 38]. Since juvenile great tits disperse during late summer-early autumn, the impact of the current environment on first-year breeders may be small compared to the older great tits which have strong site fidelity. Indeed, the association of host density manipulation was stronger among the older great tits for infection with P. circumflexum. In contrast to our prediction, habitat quality and spring date did not explain much (or any) variation in infection patterns in either of the two morphospecies.
Avian malaria, life-history and oxidative stress
There was no overall important life-history or oxidative stress parameter that explained variation in infection of both morphospecies. For P. relictum infection, age was one of the best predictors and in accordance to our prediction; older great tits were to a greater extent infected compared to younger great tits. However, there was no sign of an age effect on P. circumflexum infection, which is possibly due to the strong influence of host density (see above). Naturally, the probability of getting bitten by an infected vector increases with time regardless of species . Despite that, older individuals may have an acquired immune defence that is familiar to Plasmodium parasites, so an overall decline in cellular mechanisms and resistance with age can be an explanation for the influence of age . Indeed, since most birds are likely to have a relapse infection, the low tGSH in old P. relictum infected birds can be a result of a more rapid decline of the antioxidant defences (or indirectly via degradation of other cellular mechanisms affecting GSH synthesis) compared to uninfected ageing birds. For first year breeders, tGSH was higher when infected compared to those that were not infected, which is what we predicted during a chronic malaria infection. Alternatively, young and old individuals have different hormetic responses to parasites  or selective disappearance of the old individuals with a high tGSH in response to infection. The cause for this age difference in tGSH in response to infection warrants further investigation.
Given the difference between the sexes in behaviour and physiology during the breeding season, it was not surprising that sex explained some variation in P.relictum infection . Generally, in both mammals and birds, males are to a greater extent infected by malaria, but in the present population females have slightly higher presence of infection, but not significantly. Similarly, in a nearby forest, Wytham woods, female blue tits showed a significantly higher presence of infection of P. relictum. Possibly, the direction the association is a result of the timing of sampling i.e., breeding season (discussed below). Furthermore, there was an interaction between sex and tGSH, revealing that infected males have a higher tGSH compared to uninfected males, whereas infected females have lower (or no difference) tGSH compared to uninfected females. Interestingly, sex shows a similar pattern to age with regards to the interaction (see above, and Figure 2). The group with relatively lower presence of infection (males and young) had higher tGSH when infected compared to the uninfected birds for the same group, whereas the group with relatively higher presence of infection (females and older) had lower tGSH when infected. This sex- and age-specific response of tGSH to P. relictum parasites may play a role in susceptibility to this infection.
Alternatively, and independently of the similar age pattern, the investment into reproduction by infected females is associated with a greater physiological costs compared to uninfected females and compared to males, thus, females may not be able to up-regulate their antioxidant system . Recently, a study of great tits revealed that increasing brood size results in an increase in malaria parasitaemia in males, but that both sexes show a decrease in resistance to oxidative stress i.e., red blood cell resistance to an external free radical attack . However, a direct link between oxidative stress and parasitaemia was not found. Here clutch size was an important predictor of P. relictum infection, with uninfected birds having larger clutch size than infected birds, supporting the previously found fitness cost . ROM was slightly higher in the uninfected birds, thus the production of hydroperoxyl radicals when exposed to oxidizing agent is less likely in P. relictum prevalent birds. This seem to be in contrast to overall parasitaemia, where ROM increases with parasite density (p = 0.018), when analysed separately per species only a trend was found (P. relitum: p = 0.12, P. circumflexum: p = 0.11). As mentioned above, the overall effect of parasitaemia may be independent of parasite species virulence but rather a result of parasite metabolism . When a parasite digests haemoglobin it releases haem with iron in its ferrous state (Fe2+). This state is highly reactive and can easily oxidize to Fe3+ and thereby generate ROM [43–45]. Thus, increased parasite metabolism and abundance may be followed by increased oxidative damage (due to increased generation of hydroperoxyl radicals) unless the antioxidant defence is adequate. In a recent study of Seychelles warbler (Acrocephalus sechellensis), malaria infection was linked to a higher ROM, but parasitaemia was not measured . An alternative explanation for the patterns in both Seychelles warblers and great tits is that high generation of hydroperoxyl radicals may be linked to other unmeasured abiotic or biotic factors that increase the susceptibility to generate them, resulting in a positive association between avian malaria and ROM.
In contrast to P. relictum, GSSG (oxidized GSH) rather than the tGSH explained variation in P. circumflexum prevalence. Generally, a high GSSG indicates that the cell experiences an oxidative challenge, and that the GSH antioxidant system is in action . However, in an infected cell the antioxidant efficiency of GSH has been shown to be suppressed by a down-regulation of glutathione peroxidase and a low GSSG can be detected even though the oxidative challenge is high , see also . This is perhaps the most likely interpretation of the lower GSSG in P. circumflexum prevalent birds. However, it should be noted that in the present study all erythrocytes were measured i.e. not only the infected ones. Thus, the redox environment for the parasite may be different to the average host cell GSH/GSSG redox homeostasis. Possibly, the individuals with low GSSG are more susceptible to P. cirumflexum infection or there has been selective disappearance of individuals with high intracellular GSSG independent of infection. In captivity, healthy partridges GSSG show a quadratic relationship with age . Unfortunately, the present study is not able to disentangle the effect of age and infection on GSSG and GSH. The species difference can be a result of different manipulations of the host GSH system, parasite metabolism or virulence [14, 50, 51].